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Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 A, revealing the binding site and mechanism of action for this class of antagonists.

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not always achievable and cryoEM provides an alternative means to support structure-based ligand design. When dealing with a large number of distinct projects, and within each project a potentially large number of ligand-protein co-structures, accurate record keeping rapidly becomes challenging. Many experimental parameters are tuned for each target, including at the sample preparation, grid preparation, and microscopy stages. Therefore, accurate record keeping can be crucially important to enable long-term reproducibility, and to facilitate efficient teamwork, especially when steps of the cryoEM workflow are performed by different operators. To help deal with this challenge, we developed a web-based information management system for cryoEM, called gP2S. The application keeps track of each experiment, from sample to final atomic model, in the context of projects, a list of which is maintained in the application, or externally in a separate system. User-defined controlled vocabularies of consumables, equipment, protocols and software help describe each step of the cryoEM workflow in a structured manner. gP2S is widely configurable and, depending on the team's needs, may exist as a standalone product or be a part of a broader ecosystem of scientific applications, integrating via REST APIs with project management tools, applications tracking the production of proteins or of small molecules ligands, or applications automating data collection and storage. Users can register details of each grid and microscopy session including key experimental metadata and parameter values, and the lineage of each experimental artifact (sample, grid, microscopy session, map, etc.) is recorded. gP2S serves as a cryoEM experimental workflow organizer that enables accurate record keeping for teams, and is available under an open-source license.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Constrained peptides (CPs) have emerged as attractive candidates for drug discovery and development. To fully unlock the therapeutic potential of CPs, it is crucial to understand their physical stability and minimize the formation of aggregates that could induce immune responses. Although amyloid like aggregates have been researched extensively, few studies have focused on aggregates from other peptide scaffolds (e.g., CPs). In this work, a streamlined approach to effectively profile the nature and formation pathway of CP aggregates was demonstrated. Aggregates of various sizes were detected and shown to be amorphous. Though no major changes were found in peptide structure upon aggregation, these aggregates appeared to have mixed natures, consisting of primarily non-covalent aggregates with a low level of covalent species. This co-existence phenomenon was also supported by two kinetic pathways observed in time- and temperature-dependent aggregation studies. Furthermore, a stability study with 8 additional peptide variants exhibited good correlation between aggregation propensity and peptide hydrophobicity. Therefore, a dual aggregation pathway was proposed, with the non-covalent aggregates driven by hydrophobic interactions, whereas the covalent ones formed through disulfide scrambling. Overall, the workflow presented here provides a powerful strategy for comprehensive characterization of peptide aggregates and understanding their mechanisms of formation.

The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.

A community-wide challenge yields recommendations for improving cryo-EM structure validation.

Clustering for the kill Cluster of differentiation 20 (CD20) is a membrane protein that defines most B cell populations and is the target of therapeutic antibodies to treat malignancies and autoimmune disorders. Rougé et al. present the structure of CD20 bound to the antibody rituximab that activates the complement system to kill B cells. CD20 forms a dimer and each monomer binds one rituximab antigen-binding fragment (Fab) to give 2:2 stoichiometry. The compact packing between Fab arms and CD20 gives rise to circular assemblies with a diameter similar to that of antibody hexamers known to recruit the first component of the complement cascade. Science , this issue p. 1224

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid‐Polyamine‐organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo‐electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo‐EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all‐atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward‐facing conformation. Comparisons of the resulting Bor1p model with the X‐ray structure of AE1 in an outward‐facing conformation, together with MD simulations of inward‐facing and outward‐facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking‐bundle transport mechanism described for other members of the APC superfamily.

Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFβR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFβR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFβR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.

How activation leads to gating Voltage-gated sodium (Na v ) channels are key players in electrical signaling. Central to their function is fast inactivation, and mutants that impede this cause conditions such as epilepsy and pain syndromes. The channels have four voltage-sensing domains (VSDs), with VSD4 playing an important role in fast inactivation. Clairfeuille et al. determined the structures of a chimera in which VSD4 of the cockroach channel Na v PaS is replaced with VSD4 from human Na v 1.7, both in the apo state and bound to a scorpion toxin that impedes fast activation (see the Perspective by Chowdhury and Chanda). The toxin traps VSD4 in a deactivated state. Comparison with the apo structure shows how interactions between VSD4 and the carboxyl-terminal region change as VSD4 activates and suggests how this would lead to fast inactivation. Science , this issue p. eaav8573 ; see also p. 1278

Fast inactivation of voltage-gated sodium (Na

The caspase recruitment domain–containing protein 9 (CARD9)–B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)–CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn(2+) with picomolar affinity—a concentration comparable with the levels of readily accessible Zn(2+) in the cytosol. NMR solution structures of the CARD9–CARD in the apo and Zn(2+)-bound states revealed that Zn(2+) has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn(2+) binding, with a concomitant reduction in conformational flexibility. Moreover, Zn(2+) binding inhibited polymerization of the CARD9–CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9–CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9–Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn(2+)-mediated signaling that affects Bcl10 polymerization in innate immune responses.

We have developed new open-source software called cisTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging. cisTEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200k - 300k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments. cisTEM is available for download from cistem.org.

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001, eLife digest A molecule called adenosine triphosphate (ATP) is the energy currency in cells. Most of the ATP used by cells is made by the membrane-embedded enzyme ATP synthase. This enzyme is found in membranes inside specialized compartments known as mitochondria. ATP synthase is made up of many protein subunits that work together as a molecular machine. Hydrogen ions flow across the membrane through the ATP synthase, turning a rotor structure within the enzyme, which leads to the production of ATP. It is not known how the transport of hydrogen ions causes rotation of the rotor. Some researchers have proposed that the enzyme works as a ratchet that is driven by the random Brownian motion of the rotor. That is, the rotational position of the rotor fluctuates randomly, but a ratchet mechanism ensures that there is a net rotation in one direction. However, there is currently little experimental evidence to back up this theory, which is known as the Brownian ratchet model. Zhou, Rohou et al. used a technique called electron cryomicroscopy (or cryo-EM) to study ATP synthase from cows. The cryo-EM data made it possible to use computer software to construct a three-dimensional model of the enzyme that is more detailed than previous attempts. Zhou, Rohou et al. show that the structure of ATP synthase is flexible, with the different protein subunits bending, flexing, and rotating relative to each other. This variability in the position of the rotor is consistent with the Brownian ratchet model. Together, these findings reveal important new details about the structure of ATP synthase and provide some of the first experimental evidence for the Brownian ratchet model. The new three-dimensional structure of ATP synthase will open the door to testing hypotheses of how the ATP synthase works. DOI: http://dx.doi.org/10.7554/eLife.10180.002

Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V(1) region drives proton translocation through the membrane-embedded V(O) region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase(1–5). We induced dissociation and auto-inhibition of the V(1) and V(O) regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae(6,7), allowing us to obtain a ~3.9-Å resolution electron cryomicroscopy map of the V(O) complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac(8)c′c″de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c″ interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

The biological effects of Latrodectus spider venom are similar in animals from different phyla, but these symptoms are caused by distinct phylum-specific neurotoxins (collectively called latrotoxins) with molecular masses ranging from 110 to 140 kDa. To date, the venom has been found to contain five insecticidal toxins, termed alpha, beta, gamma, delta and epsilon-latroinsectotoxins (LITs). There is also a vertebrate-specific neurotoxin, alpha-latrotoxin (alpha-LTX), and one toxin affecting crustaceans, alpha-latrocrustatoxin (alpha-LCT). These toxins stimulate massive release of neurotransmitters from nerve terminals and act (1) by binding to specific receptors, some of which mediate an exocytotic signal, and (2) by inserting themselves into the membrane and forming ion-permeable pores. Specific receptors for LITs have yet to be identified, but all three classes of vertebrate receptors known to bind alpha-LTX are also present in insects. All LTXs whose structures have been elucidated (alpha-LIT, delta-LIT, alpha-LTX and alpha-LCT) are highly homologous and have a similar domain architecture, which consists of a unique N-terminal sequence and a large domain composed of 13-22 ankyrin repeats. Three-dimensional (3D) structure analysis, so far done for alpha-LTX only, has revealed its dimeric nature and an ability to form symmetrical tetramers, a feature probably common to all LTXs. Only tetramers have been observed to insert into membranes and form pores. A preliminary 3D reconstruction of a delta-LIT monomer demonstrates the spatial similarity of this toxin to the monomer of alpha-LTX.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of β-amyloid (Aβ) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aβ fibrillation is extensively documented, much is still unknown about the formation of these Aβ aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aβ(1-42), a particularly pathogenic variant of Aβ peptide. Our model reveals that the individual layers of the Aβ fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal β-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aβ peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aβ peptide.

CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope?s contrast transfer function (CTF) to an image?s amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.

The structures of many helical protein filaments can be derived from electron micrographs of their suspensions in thin films of vitrified aqueous solutions. The most successful and generally-applicable approach treats short segments of these filaments as independent “single particles”, yielding near-atomic resolution for rigid and well-ordered filaments. The single-particle approach can also accommodate filament deformations, yielding sub-nanometer resolution for more flexible filaments. However, in the case of thin and flexible filaments, such as some amyloid-β (Aβ) fibrils, the single-particle approach may fail because helical segments can be curved or otherwise distorted and their alignment can be inaccurate due to low contrast in the micrographs. We developed new software called Frealix that allows the use of arbitrarily short filament segments during alignment to approximate even high curvatures. All segments in a filament are aligned simultaneously with constraints that ensure that they connect to each other in space to form a continuous helical structure. In this paper, we describe the algorithm and benchmark it against datasets of Aβ(1–40) fibrils and tobacco mosaic virus (TMV), both analyzed in earlier work. In the case of TMV, our algorithm achieves similar results to single-particle analysis. In the case of Aβ(1–40) fibrils, we match the previously-obtained resolution but we are also able to obtain reliable alignments and ∼8-A reconstructions from curved filaments. Our algorithm also offers a detailed characterization of filament deformations in three dimensions and enables a critical evaluation of the worm-like chain model for biological filaments.

The traditional tools of the structural biologist seeking to understand macromolecules and their complexes are X-ray crystallography and NMR spectroscopy. Single-particle cryo-electron microscopy (cryo-EM) has established itself as a new structural-biology technique over the last 15 years. Spectacular insights into the functioning of macromolecular complexes have been achieved especially from combining cryo-EM with the earlier approaches. The resolution levels achieved improved over the last decade from ∼10 A to sometimes better than ∼4 A, meaning that a de novo structure determination based on single-particle cryo-EM studies alone is now feasible. More challenging is the new perspective that cryo-EM brings: sorting heterogeneous populations of molecules into individual three-dimensional conformers resulting in sequences of related three-dimensional structures, or in short ‘4D cryo-EM’. Thanks to these developments, single-particle cryo-EM has become the technique of choice for shedding light on the functioning of many a complex biological system. The design of the software instrumentation for 4D cryo-EM is crucial. In this chapter we elaborate on organization issues for single-particle cryo-EM software, as exemplified by recent developments in the IMAGIC 4D software system. Keywords: cryo-electron microscopy; IMAGIC 4D

A commonly used strategy by microorganisms to survive multiple stresses involves a signal transduction cascade that increases the expression of stress-responsive genes. Stress signals can be integrated by a multiprotein signaling hub that responds to various signals to effect a single outcome. We obtained a medium-resolution cryo–electron microscopy reconstruction of the 1.8-megadalton “stressosome” from Bacillus subtilis . Fitting known crystal structures of components into this reconstruction gave a pseudoatomic structure, which had a virus capsid–like core with sensory extensions. We suggest that the different sensory extensions respond to different signals, whereas the conserved domains in the core integrate the varied signals. The architecture of the stressosome provides the potential for cooperativity, suggesting that the response could be tuned dependent on the magnitude of chemophysical insult.

α-Latrotoxin (α-LTX) from black widow spider venom induces exhaustive release of neurotransmitters from vertebrate nerve terminals and endocrine cells. This 130-kDa protein has been employed for many years as a molecular tool to study exocytosis. However, its action is complex: in neurons, α-LTX induces massive secretion both in the presence of extracellular Ca2+ (Ca2+e) and in its absence; in endocrine cells, it usually requires Ca2+e. To use this toxin for further dissection of secretory mechanisms, one needs an in-depth understanding of its functions. One such function that explains some α-LTX effects is its ability to form cation-permeable channels in artificial lipid bilayers. The mechanism of α-LTX pore formation, revealed by cryo-electron microscopy, involves toxin assembly into homotetrameric complexes which harbour a central channel and can insert into lipid membranes. However, in biological membranes, α-LTX cannot exert its actions without binding to specific receptors of the plasma membrane. Three proteins with distinct structures have been found to bind α-LTX: neurexin Iα, latrophilin 1 and receptor-like protein tyrosine phosphatase σ. Upon binding a receptor, α-LTX forms channels permeable to cations and small molecules; the toxin may also activate the receptor. To distinguish between the pore- and receptor-mediated effects, and to study structure-function relationships in the toxin, α-LTX mutants have been used. At least one non-pore-forming α-LTX mutant can activate latrophilin, a G protein-coupled receptor, causing release of Ca2+ from intracellular stores. Latrophilin action still requires Ca2+e and may trigger transmitter secretion either by itself or by activating Ca2+-channels and/or inducing Ca2+ waves. These results reveal two Ca2+e-dependent mechanisms of α-LTX action (membrane pore formation and signalling via latrophilin), but how α-LTX triggers Ca2+e-independent neurotransmitter release still remains unexplained. Hypotheses for this action include direct interaction with intracellular components involved in exocytosis or the effects of α-LTX pores.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.